CN112715351B - Rapid breeding method of phellinus igniarius - Google Patents
Rapid breeding method of phellinus igniarius Download PDFInfo
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Images
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Mushroom Cultivation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a rapid breeding method of phellinus igniarius, which takes phellinus igniarius and ganoderma applanatum as two hybrid parents for cultivation. According to the invention, the low-yield and high-drug-effect phellinus igniarius and the high-yield ganoderma applanatum are used as two hybrid parents, so that the superior characters in ganoderma applanatum are integrated and expressed in phellinus igniarius, and the yield and the quality of phellinus igniarius are improved.
Description
Technical Field
The invention relates to the technical field of phellinus igniarius breeding, in particular to a rapid breeding method of phellinus igniarius.
Background
Phellinus igniarius (Phellinus igniarius)Pellinus igniarius) Also called Morus alba, Phellinus genus of Aphyllophorales (Polyporaceae) of Hymenomycetes (Hymenomycetes) of Basidiomycotina (Basidio-mycotina) (Phellinus)Pellinus). In China, the application of phellinus igniarius has been used for more than two thousand years. The earliest record of medicine can be traced back to the descriptions of the medicinal properties of the phellinus linteus, such as benefiting the five internal organs, ventilating the intestines and stomach and expelling the toxic gas, in the classic 'Shennong' herbal classic of Chinese medicine in the Han Dynasty and 'the compendium of materia Medica' in the Ming Dynasty. The ingredients, pharmacology and compound recipe of Phellinus linteus are recorded in Chinese medicine dictionary, Chinese materia medica and Chinese pharmacopoeia. Phellinus linteus is a rare medicinal fungus, and it is resistant toThe cancer effect is better than that of ganoderma lucidum, agaricus blazei and the like, and the cancer is called as 'forest gold'. At present, phellinus igniarius is a fungus with the highest efficiency in the internationally recognized biological cancer treatment field, and is now a hot spot for research and development in the fields of health food and medicine at home and abroad.
Phellinus igniarius has high medicinal value, and the application of Phellinus igniarius in cancer treatment is more and more emphasized. Phellinus linteus contains polysaccharides, flavonoids, polyphenols, terpenes, sterols and other medicinal active ingredients, and is sweet, pungent, mild and nontoxic in nature. In Shenghui Fang, it is stated that Phellinus Linteus can treat female leukorrhea with reddish discharge, blood disease, abdominal mass, swelling and pain, yin-yang cold and heat, infertility and irregular menstruation. Modern researches show that phellinus igniarius also has the effects of resisting tumors, resisting oxidation, reducing blood sugar and resisting inflammation, and also has the effects of protecting liver, resisting cirrhosis, treating gout, stopping bleeding, promoting blood circulation, resisting viruses, resisting aging, enhancing the immunity of organisms and the like. Phellinus linteus, as a medicinal material effective for the treatment of cancer, has been studied very early in Japan and Korea and is listed as an anticancer drug in the national drug code. In the second war, cancer cases were greatly reduced by residents in Kawasaki areas taking Phellinus linteus, which is abundant in the island, and thereafter, Phellinus linteus began to grow slowly in Japan, Korea, Taiwan of China, and the like. Ikekawa T was the first to initiate modern medical research at Phellinus linteus, and suggests the antitumor effects of some basidiomycetes, particularly sang huang. In 1968, clinical experiments of antitumor of phellinus linteus by the department of chemotherapy of the national institute of tumor in Japan found that the tumor inhibition rate of phellinus linteus is as high as 96.7%, and the phellinus linteus has no side effect after being eaten for a long time. By means of inhibition studies on AKT signals, researchers found that Phellinus linteus inhibited growth, angiogenesis and invasive behavior of breast cancer cells. The experimental result also shows that the phellinus igniarius can improve the activation degree of immune cells by 2-129 times. In addition, Phellinus Linteus is a fungus with abundant nutrition, and contains various nutritional components such as polysaccharide, amino acid, larch acid, veratrine, mushroom acid, and m-dimethoxyphthalic acid.
However, since Phellinus linteus group fungi are restricted by the particularity and complexity of their physiological ecology, and the natural fruiting bodies formed in nature grow slowly and have a low survival rate, the present invention intends to study a method for rapidly cultivating high-yield Phellinus linteus, so that the supply of Phellinus linteus can be greatly increased.
Disclosure of Invention
The invention discloses a rapid breeding method of phellinus igniarius, which takes low-yield and high-drug-effect phellinus igniarius and high-yield ganoderma applanatum as two hybrid parents so that the advantageous characters in ganoderma applanatum are integrated and expressed in phellinus igniarius.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a rapid breeding method of Phellinus igniarius uses Phellinus igniarius and Ganoderma Applanatum as two hybrid parents for cultivation.
Further, the invention provides a specific step of culturing two hybrid parents by taking phellinus igniarius and ganoderma applanatum as the parents, which specifically comprises the following steps:
s1, cleaning phellinus igniarius and Ganoderma Applanatum fruiting bodies, and respectively cutting two kinds of mushroom meat in a superclean bench by using a sterilization knife to put the two kinds of mushroom meat in a mushroom meat culture medium for culturing;
s2, selecting the hyphae cultured in the step S1 to a hypha culture medium for co-culture, wherein the inoculation distance between phellinus igniarius hyphae and Ganoderma applanatum hyphae is 1.2-2.5 cm; and culturing at room temperature for 22-25 h, then sequentially adding 0.2-0.4 mg/mL calcium chloride solution and 0.8-1.2 mol/L transfer factor extracting solution, wherein 1-5 drops of the calcium chloride solution and 0.8-1.5 mL of the transfer factor extracting solution are added to each 100mL of the culture medium, transferring to a constant temperature environment of 36-40 ℃ for culturing for 32-38 h, then transferring to a constant temperature environment of 10-14 ℃ for further culturing for 3-6 h, finally culturing at room temperature for 10-14 h, and selecting fused mycelia to inoculate a sterilized culture material bag for producing a new phellinus igniarius sporophore.
Preferably, the mushroom meat culture medium consists of the following components in parts by weight: 180-250 parts of potatoes, 8-20 parts of peach wood chip powder, 8-20 parts of mulberry twig wood chip powder, 18-22 parts of glucose, 15-20 parts of agar and 800-1000 parts of water; the preparation method of the mushroom culture medium comprises the following steps: adding water into potato, peach wood chip powder and mulberry branch wood chip powder, boiling for 10-20 min, filtering out the peach wood chip powder and the mulberry branch wood chip powder, heating the filtrate, glucose and agar together until the filtrate is dissolved, and finally adjusting the pH value to 6.2-7.0. More preferably, the mushroom meat culture medium consists of the following components in parts by weight: 200 parts of potato, 10 parts of peach wood chip powder, 10 parts of mulberry twig wood chip powder, 20 parts of glucose, 18 parts of agar and 1000 parts of water, and the pH value is adjusted to 6.5.
Preferably, the components and the preparation method of the hypha culture medium are the same as those of the mushroom culture medium.
Preferably, the culture material bag comprises the following components in parts by weight: 1-3 parts of peach wood chip powder, 1-5 parts of mulberry twig wood chip powder, 1-3 parts of bagasse, 1-2 parts of wheat bran, 1-2 parts of corn flour and 1-2 parts of agar, mixing, adding water, stirring until the mixture is moist, bagging and sterilizing. More preferably, the culture material bag comprises the following components in parts by weight: 2 parts of peach wood chip powder, 3 parts of mulberry twig wood chip powder, 2 parts of bagasse, 1 part of wheat bran, 1 part of corn flour and 1 part of agar, mixing, adding water, stirring until the mixture has a moist feeling, bagging and sterilizing.
Preferably, in the step S2, the inoculation distance is 2 cm.
Preferably, in step S2, the specific process of culturing includes: after culturing for 24h at room temperature, sequentially adding 0.3mg/mL calcium chloride solution and 1.0mol/L transfer factor extracting solution, wherein 2 drops of the calcium chloride solution and 1mL of the transfer factor extracting solution are added to each 100mL of culture medium, transferring to a constant temperature environment of 38 ℃ for culturing for 36h, then transferring to a constant temperature environment of 12 ℃ for continuous culturing for 4h, and finally culturing at room temperature for 12 h.
Preferably, the constant-temperature environment at the temperature of 36-40 ℃ is a constant-temperature incubator; the constant temperature environment of 10-14 ℃ is a refrigerator.
The transfer factor extracting solution is prepared from spleen of healthy animal, and is extracted from solution containing polypeptide, amino acid and polynucleotide mixture according to standard number WS1- (X-451) -2003Z.
According to the rapid breeding method of the phellinus igniarius, the phellinus igniarius with low yield and high drug effect and the ganoderma applanatum with high yield are used as two hybrid parents, so that the advantageous characters in the ganoderma applanatum are integrated and expressed in the phellinus igniarius, and the yield of the phellinus igniarius is improved. Meanwhile, the invention further adopts a hypha fusion method to culture new species, so that the breeding time of the large fungi can be greatly shortened.
Furthermore, in the hypha culture process, calcium ions with proper concentration are added and matched with a certain temperature difference, so that the rapid fusion of cells is promoted. In addition, the invention adds host wood chip components suitable for the growth of phellinus igniarius and ganoderma applanatum into the mushroom culture medium, so that the two fungi can better grow in the culture medium.
Detecting non-target metabolites of the phellinus igniarius-lingua fungi by a liquid chromatography-tandem triple quadrupole time-of-flight high resolution mass spectrometry method, wherein enrichment analysis results show that: compared with phellinus igniarius sporocarp, the activity of 10 substances in related substances of carbon metabolism is obviously improved, the activity of 25 substances in an ATP binding cassette transporter family is obviously improved, and the activity of 13 substances in flavonoid biosynthesis is obviously improved, so that the phellinus igniarius-Ganoderma Applanatum cultured innovatively is active in metabolism and vigorous in growth, and is beneficial to accumulation of carbohydrate and flavonoid substances. The accumulation of carbohydrate is an important and key index for improving the yield, the flavonoid is an important category for embodying the drug effect of the phellinus igniarius, and the comprehensive detection result analysis of the fruiting bodies, hyphae and metabolites of the new species of phellinus igniarius-artist's tongue fungus and the original species of phellinus igniarius shows that the yield and the quality of the new breed are improved.
Drawings
FIG. 1 shows the microscopic morphology of Ganoderma Applanatum cultured mycelia.
FIG. 2 shows the microscopic morphology of cultured Phellinus linteus mycelia.
FIG. 3 shows the mycelium microscopic morphology of Phellinus linteus-Ganoderma Applanatum coculture.
Detailed Description
The present invention is further illustrated by the following specific examples, but the scope of the present invention is not limited to the following examples.
The material acquisition mode is as follows: harvesting Phellinus igniarius sporophore from wild Morus alba in one hundred thousand mountains, and storing at low temperature (about 4 deg.C) in a field sampling box; collecting Ganoderma Applanatum fruiting body from artificially cultured peach tree in Dazhong of Nanning institute, placing into a field sampling box, and storing at low temperature (about 4 deg.C) for taking back.
Example 1
A rapid breeding method of phellinus igniarius comprises the following steps:
s1, cleaning the surface dust and other impurities of phellinus igniarius and Ganoderma Applanatum fruiting body, washing twice with 75% alcohol, respectively cutting two kinds of fungus meat with the size of mung bean into fungus meat culture medium with a sterilization knife in a super clean workbench, and culturing;
the main components of the mushroom meat culture medium are as follows: 200g of potato, 20g of glucose, 18g of agar, 1000g of water, 10g of peach wood chip powder and 10g of mulberry twig wood chip powder, and adjusting the pH value to 6.5. The preparation process comprises the following steps: cleaning and peeling potatoes, cutting the potatoes into small pieces, adding peach wood chip powder and mulberry twig wood chip powder, adding water to boil the potatoes thoroughly (boiling the potatoes for 3min with strong fire and then boiling the potatoes for 10min with small fire), filtering the potatoes by using eight layers of gauze, filtering the peach wood chip powder and the mulberry twig wood chip powder, adding agar and glucose into filtrate, continuously heating and stirring the mixture uniformly, finally adjusting the pH value to 6.5, subpackaging the mixture into a test tube or a conical flask, adding a plug and wrapping the mixture, sterilizing the mixture for about 30 minutes at the temperature of 115 ℃, cooling and storing the mixture for later use.
S2, picking the hyphae cultured in the step S1 into a hypha culture medium for co-culture, wherein the inoculation distance between phellinus igniarius hyphae and Ganoderma applanatum hyphae is 2 cm; after 24h of culture, adding 2 drops of 0.3mg/mL calcium chloride solution and 1mL transfer factor extracting solution with the concentration of 1mol/L into each 100mL of culture medium, transferring the mixture into a constant-temperature incubator at 38 ℃ for culture for 36h, transferring the mixture into a refrigerator at 12 ℃ for continuous culture for 4h, finally culturing the mixture at room temperature for 12h, and selecting fused hypha to inoculate a sterilized culture material bag for producing a new variety of phellinus igniarius sporocarp.
The main components of the culture material bag are as follows: the preparation method comprises the steps of mixing 20g of peach wood chip powder, 30g of mulberry twig wood chip powder, 20g of bagasse, 10g of wheat bran, 10g of corn flour and 10g of agar, adding water, stirring until the mixture is moist, bagging and sterilizing for later use.
Example 2
A rapid breeding method of phellinus igniarius comprises the following steps:
s1, cleaning the surface dust and other impurities of phellinus igniarius and Ganoderma Applanatum fruiting body, washing twice with 75% alcohol, respectively cutting two kinds of fungus meat with the size of mung bean into fungus meat culture medium with a sterilization knife in a super clean workbench, and culturing;
the main components of the mushroom meat culture medium are as follows: 220g of potatoes, 22g of glucose, 20g of agar, 1000g of water, 20g of peach wood chip powder and 20g of mulberry twig wood chip powder, and adjusting the pH value to 6.5. The preparation process comprises the following steps: cleaning and peeling potatoes, cutting the potatoes into small pieces, adding peach wood chip powder and mulberry twig wood chip powder, adding water to boil the potatoes thoroughly (boiling the potatoes for 3min with strong fire and then boiling the potatoes for 15min with small fire), filtering the potatoes by using eight layers of gauze, filtering the peach wood chip powder and the mulberry twig wood chip powder, adding agar and glucose into filtrate, continuously heating and stirring the mixture uniformly, finally adjusting the pH value to 6.8, subpackaging the mixture into a test tube or a conical flask, adding a plug and wrapping the mixture, sterilizing the mixture for about 30 minutes at the temperature of 115 ℃, cooling and storing the mixture for later use.
S2, picking the hyphae cultured in the step S1 into a hypha culture medium for co-culture, wherein the inoculation distance between phellinus igniarius hyphae and Ganoderma applanatum hyphae is 2.3 cm; after 24h of culture, adding 3 drops of 0.2mg/mL calcium chloride solution and 1.2mL transfer factor extracting solution with the concentration of 0.9mol/L into each 100mL culture medium, transferring the mixture into a constant-temperature incubator at 36 ℃ for culture for 37h, transferring the mixture into a refrigerator at 10 ℃ for continuous culture for 5h, finally culturing the mixture at room temperature for 10h, and selecting fused hyphae to inoculate into a sterilized culture material bag for producing new phellinus igniarius sporocarp.
The main components of the culture material bag are as follows: 10g of peach wood chip powder, 35g of mulberry twig wood chip powder, 25g of bagasse, 12g of wheat bran, 12g of corn flour and 20g of agar, and the preparation method comprises the steps of mixing, adding water, stirring until the mixture is moist, bagging and sterilizing for later use.
Example 3
A rapid breeding method of phellinus igniarius comprises the following steps:
s1, cleaning the surface dust and other impurities of phellinus igniarius and Ganoderma Applanatum fruiting body, washing twice with 75% alcohol, respectively cutting two kinds of fungus meat with the size of mung bean into fungus meat culture medium with a sterilization knife in a super clean workbench, and culturing;
the main components of the mushroom meat culture medium are as follows: 180g of potatoes, 18g of glucose, 20g of agar, 950g of water, 12g of peach wood chip powder and 15g of mulberry twig wood chip powder, and adjusting the pH value to 6.5. The preparation process comprises the following steps: cleaning and peeling potatoes, cutting the potatoes into small pieces, adding peach wood chip powder and mulberry twig wood chip powder, adding water to boil the potatoes thoroughly (boiling the potatoes for 3min with strong fire and then boiling the potatoes for 10min with small fire), filtering the potatoes by using eight layers of gauze, filtering the peach wood chip powder and the mulberry twig wood chip powder, adding agar and glucose into filtrate, continuously heating and stirring the mixture uniformly, finally adjusting the pH value to 6.3, subpackaging the mixture into a test tube or a conical flask, adding a plug and wrapping the mixture, sterilizing the mixture for about 30 minutes at the temperature of 115 ℃, cooling and storing the mixture for later use.
S2, picking the hyphae cultured in the step S1 into a hypha culture medium for co-culture, wherein the inoculation distance between phellinus igniarius hyphae and Ganoderma applanatum hyphae is 2 cm; after 24h of culture, adding 2 drops of 0.3mg/mL calcium chloride solution and 1mL transfer factor extracting solution with the concentration of 1mol/L into each 100mL of culture medium, transferring the mixture into a constant-temperature incubator at 38 ℃ for culture for 36h, transferring the mixture into a refrigerator at 12 ℃ for continuous culture for 4h, finally culturing the mixture at room temperature for 12h, and selecting fused hypha to inoculate a sterilized culture material bag for producing a new variety of phellinus igniarius sporocarp.
The main components of the culture material bag are as follows: 30g of peach wood chip powder, 10g of mulberry twig wood chip powder, 20g of bagasse, 10g of wheat bran, 10g of corn flour and 20g of agar.
Comparative example 1
A method for breeding phellinus igniarius comprises the following steps:
s1, cleaning impurities such as dust on the surface of phellinus igniarius, washing twice with 75% alcohol, cutting mung bean-sized mushroom meat into mushroom meat culture media respectively by using a sterilization knife in a super clean workbench, and culturing;
the main components of the mushroom meat culture medium are as follows: 200g of potato, 20g of glucose, 18g of agar, distilled water, 10g of peach wood chip powder and 10g of mulberry twig wood chip powder, and adjusting the pH value to 6.5. The preparation process comprises the following steps: firstly, cleaning and peeling potatoes, weighing 200g of potatoes, cutting the potatoes into small pieces, adding water, boiling the small pieces thoroughly (boiling for 20-30 minutes and being scattered by a glass rod), filtering the small pieces by eight layers of gauze, heating, adding 18g of agar, continuously heating, stirring and uniformly mixing, adding 20g of glucose, 10g of peach wood chip powder and mulberry twig wood chip powder respectively after the agar is dissolved, uniformly stirring, slightly cooling, adding distilled water to supplement water to 1100g, subpackaging test tubes or conical bottles, plugging and binding, sterilizing at 115 ℃ for about 30 minutes, cooling and storing for later use.
S2, picking the hyphae cultured in the step S1 into a hypha culture medium for culturing, adding 2 drops of 0.3mg/mL calcium chloride solution and 1mL transfer factor extracting solution with the concentration of 1mol/L into each 100mL culture medium after culturing for 24h, transferring into a constant-temperature incubator at 38 ℃ for culturing for 36h, then transferring into a refrigerator at 12 ℃ for continuous culturing for 4h, finally culturing at room temperature for 12h, picking the fused hyphae and inoculating into a sterilized culture material bag for producing phellinus igniarius sporocarp.
The main components of the culture material bag are as follows: the preparation method comprises the steps of mixing 20g of peach wood chip powder, 30g of mulberry twig wood chip powder, 20g of bagasse, 10g of wheat bran, 10g of corn flour and 10g of agar, adding water, stirring until the mixture is moist, bagging and sterilizing for later use.
Comparative example 2
A breeding method of Ganoderma applanatum comprises the following steps:
s1, cleaning the ganoderma applanatum to remove impurities such as dust on the surface, washing twice with 75% alcohol, cutting mung bean-sized mushroom meat in a super clean bench with a sterilizing knife, and culturing in a mushroom meat culture medium;
the main components of the mushroom meat culture medium are as follows: 200g of potato, 20g of glucose, 18g of agar, distilled water, 10g of peach wood chip powder and 10g of mulberry twig wood chip powder, and adjusting the pH value to 6.5. The preparation process comprises the following steps: firstly, cleaning and peeling potatoes, weighing 200g of potatoes, cutting the potatoes into small pieces, adding water, boiling the small pieces thoroughly (boiling for 20-30 minutes and being scattered by a glass rod), filtering the small pieces by eight layers of gauze, heating, adding 18g of agar, continuously heating, stirring and uniformly mixing, adding 20g of glucose, 10g of peach wood chip powder and mulberry twig wood chip powder respectively after the agar is dissolved, uniformly stirring, slightly cooling, adding distilled water to supplement water to 1100g, subpackaging test tubes or conical bottles, plugging and binding, sterilizing at 115 ℃ for about 30 minutes, cooling and storing for later use.
S2, selecting the hyphae cultured in the step S1 to a hypha culture medium for culturing, adding 2 drops of 0.3mg/mL calcium chloride solution and 1mL transfer factor extracting solution with the concentration of 1mol/L into each 100mL culture medium after culturing for 24h, transferring to a constant-temperature incubator at 38 ℃ for culturing for 36h, then transferring to a refrigerator at 12 ℃ for continuous culturing for 4h, finally culturing at room temperature for 12h, selecting the fused hyphae and inoculating to a sterilized culture material bag for producing Ganoderma applanatum sporocarp.
The main components of the culture material bag are as follows: the preparation method comprises the steps of mixing 20g of peach wood chip powder, 30g of mulberry twig wood chip powder, 20g of bagasse, 10g of wheat bran, 10g of corn flour and 10g of agar, adding water, stirring until the mixture is moist, bagging and sterilizing for later use.
Comparative example 3
Comparative example 3 is essentially the same as example 1, but in example 2:
after 24 hours of culture, 2 drops of 0.3mg/mL calcium chloride solution and 1mL transfer factor extracting solution with the concentration of 1mol/L are added into each 100mL culture medium, the mixture is transferred into a constant temperature incubator with the temperature of 38 ℃ for culture for 36 hours, then the mixture is transferred into a refrigerator with the temperature of 12 ℃ for continuous culture for 4 hours, finally the mixture is cultured for 12 hours at room temperature,
replacing the steps as follows: after 24h of culture, 3 drops of 0.2mg/mL calcium chloride solution were added and the culture was continued at room temperature for 52 h.
Comparative example 4
Comparative example 4 is substantially the same as example 1, except that in this example, a calcium chloride solution and a transfer factor extract solution were not added dropwise.
Comparative example 5
Comparative example 5 is substantially the same as example 1, but in this example, the main components of the meat culture medium: 200g of potato, 20g of glucose, 18g of agar and distilled water, and the pH value is adjusted to 6.5. The preparation process comprises the following steps: the preparation method comprises the steps of firstly cleaning and peeling potatoes, weighing 200g of the potatoes, cutting the potatoes into small pieces, adding water, boiling the small pieces thoroughly (boiling for 20-30 minutes and being scattered by a glass rod), filtering the small pieces with eight layers of gauze, heating, adding 18g of agar, continuously heating, stirring and mixing uniformly, adding 20g of glucose after the agar is dissolved, stirring uniformly, slightly cooling, adding distilled water to complement water to 1080g, subpackaging the obtained product in test tubes or conical bottles, adding plugs, binding up the obtained product, sterilizing at 115 ℃ for about 30 minutes, cooling and storing the obtained product for later use.
The results of the hyphal growth and the fruiting body yield and quality of examples 1-3 and comparative examples 1-5 are shown in tables 1 and 2, wherein: the method for extracting and purifying the total flavonoids in the sporocarp comprises the following steps: collecting Phellinus Linteus50g of the powder is placed in a swing type grinder to be ground into medium powder, the medium powder is uniformly mixed, 20g of the powder is precisely weighed and placed in a round bottom flask, 400 mL of 70% ethanol is added, the mixture is soaked for 24 hours at room temperature and filtered, the residue is subjected to one time of repeated operation, two leaching solutions are combined, the ethanol is concentrated and recovered until no alcohol smell exists, 70% ethanol is added to reach a constant volume of 500mL, and the mixture is uniformly shaken for standby. Measuring total flavone content by nitrous acid-aluminum nitrate method, respectively taking 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL0.307 mg/mL rutin standard solution in 10.0 mL measuring flask, adding 0.3 mL5% NaNO2Shaking, standing for 5 min; 0.3 mL5% Al (NO)3)3Shaking, standing for 5 min; 4.0 mL of 4% NaOH, adding water to a constant volume of 10.0 mL, shaking, standing for 10min, measuring absorbance A at 504 nm, taking the mass concentration (c, mg/mL) of rutin as an abscissa and the absorbance (A) as an ordinate, and performing parallel measurement for 3 times to prepare a standard curve.
And (3) measuring the absorbance of the sample solution at 504 nm, and calculating the rutin mass concentration (c, mg/mL) in the reaction system by using a regression equation of a rutin standard curve.
Flavone content = flavone concentration in sample/flavone concentration of sample purified substance × 100%.
Testing method of hypha dry weight: and (3) taking 200mL of hypha culture medium, cleaning the culture medium, drying the hypha, weighing to obtain a weight G, and weighing the weight G/2 to obtain the dry weight of the hypha.
Measurement method of hypha growth rate: and (3) inoculating the bacterial colony on the edge of the hypha cultured in the step (2) into a new culture dish for culturing by using an aseptic puncher, recording the diameter of the bacterial colony in the new culture dish after culturing for 3 days, and calculating the length of the radius of the bacterial colony increased every day.
TABLE 1 hyphal growth
TABLE 2 yield and quality of fruiting bodies
Claims (6)
1. A rapid breeding method of phellinus igniarius is characterized by comprising the following steps: culturing two hybrid parents of phellinus igniarius and ganoderma applanatum;
the method specifically comprises the following steps:
s1, cleaning phellinus igniarius and Ganoderma Applanatum fruiting bodies, and respectively cutting two kinds of mushroom meat in a superclean bench by using a sterilization knife to put the two kinds of mushroom meat in a mushroom meat culture medium for culturing;
s2, selecting the hyphae cultured in the step S1 to a hypha culture medium for co-culture, wherein the inoculation distance between phellinus igniarius hyphae and Ganoderma applanatum hyphae is 1.2-2.5 cm; culturing at room temperature for 22-25 h, then sequentially adding 0.2-0.4 mg/mL calcium chloride solution and 0.8-1.2 mol/L transfer factor extracting solution, wherein 1-5 drops of calcium chloride solution and 0.8-1.5 mL transfer factor extracting solution are added to each 100mL culture medium, transferring to a constant temperature environment of 36-40 ℃ for culturing for 32-38 h, then transferring to a constant temperature environment of 10-14 ℃ for further culturing for 3-6 h, finally culturing at room temperature for 10-14 h, and selecting fused hypha to inoculate a sterilized culture material bag for producing a new phellinus igniarius sporocarp;
the mushroom meat culture medium comprises the following components in parts by weight: 180-250 parts of potatoes, 8-20 parts of peach wood chip powder, 8-20 parts of mulberry twig wood chip powder, 18-22 parts of glucose, 15-20 parts of agar and 800-1000 parts of water;
the preparation method of the mushroom culture medium comprises the following steps: adding water into potato, peach wood chip powder and mulberry branch wood chip powder, boiling for 10-20 min, filtering out the peach wood chip powder and the mulberry branch wood chip powder, heating the filtrate, glucose and agar together until the filtrate is dissolved, and finally adjusting the pH value to 6.2-7.0;
the components and the preparation method of the hypha culture medium and the mushroom culture medium are the same;
the culture material bag comprises the following components in parts by weight: 1-3 parts of peach wood chip powder, 1-5 parts of mulberry twig wood chip powder, 1-3 parts of bagasse, 1-2 parts of wheat bran, 1-2 parts of corn flour and 1-2 parts of agar, mixing, adding water, stirring until the mixture is moist, bagging and sterilizing;
the transfer factor extracting solution is prepared from spleen of healthy animal, and is extracted from solution containing polypeptide, amino acid and polynucleotide mixture according to standard number WS1- (X-451) -2003Z.
2. The rapid breeding method of Phellinus linteus according to claim 1, characterized in that:
in step S2, the distance of inoculation is 2 cm.
3. The rapid breeding method of Phellinus linteus according to claim 1, characterized in that:
in step S2, the specific process of culturing includes: after 24 hours of culture, adding 0.3mg/mL calcium chloride solution and 1.0mol/L transfer factor extracting solution in turn, wherein 2 drops of calcium chloride solution and 1mL of transfer factor extracting solution are added to each 100mL of culture medium, transferring to the constant temperature environment of 38 ℃ for culture for 36 hours, then transferring to the constant temperature environment of 12 ℃ for continuous culture for 4 hours, and finally culturing at room temperature for 12 hours.
4. The rapid breeding method of Phellinus linteus according to claim 1, characterized in that:
the constant temperature environment at 36-40 ℃ is a constant temperature incubator; the constant temperature environment of 10-14 ℃ is a refrigerator.
5. The rapid breeding method of Phellinus linteus according to claim 1, characterized in that:
the mushroom meat culture medium comprises the following components in parts by weight: 200 parts of potato, 20 parts of glucose, 18 parts of agar, 1000 parts of distilled water, 10 parts of peach wood chip powder and 10 parts of mulberry twig wood chip powder, and adjusting the pH value to 6.5.
6. The rapid breeding method of Phellinus linteus according to claim 1, characterized in that:
the culture material bag comprises the following components in parts by weight: 2 parts of peach wood chip powder, 3 parts of mulberry twig wood chip powder, 2 parts of bagasse, 1 part of wheat bran, 1 part of corn flour and 1 part of agar, mixing, adding water, stirring until the mixture has a moist feeling, bagging and sterilizing.
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